Characterization of the Human Interleukin-8 Type B Receptor Interleukin-8 (IL-8), neutrophil-activating peptide 2 (NAP-2), and gro/melanoma growth-stimulatory activity (GRO) are structurally related human peptides that act as mediators of inflammation. These chemokines elicit activity subseuent to binding specific receptor molecules on the surface of responsive cells - including neutrophils. Two human neutrophil L-8 receptors have been cloned, type A (L-8RA) and type B (IL-8RB). These receptors belong to the family of G-protein-coupled, seven-transmembrane receptors. The two receptors show 32% similarity in their N-termini and 83% similarity over the remainder of the protein. Both receptors bind L-8 with high affinity, but IL 8RB also binds GRO and NAP-2, both of which can compete with IL-8 for binding. The long term objective of this study is to determine the purpose of the second IL8 receptor that also binds GRO and NAP-2. We hypothesize that the binding of different chemokines may have a regulatory function on neutrophil inflammatory responses. Our immediate goal is to define the mechanism by which IL-8. GRO. and N-.2 bind to the IL-8RB. Our first specific aim is to determine the regions of the IL-8RB necessary for binding IL-8, GRO and NAP-2. We have obtained the p3 cDNA (1.9 kb) that includes the human IL 8RB ORF from Philip Murphy at the NIH. Utilizing PCR we then isolated the 1.1 kb DNA fragment corresponding to the IL8RB ORF. We have transformed E. coli cells (XL Blue) with the plasmid l-Gl8R (Pharmacia) containing our IL8RB insert by electroporation. Restriction digestion and analysis by agarose gel electrophoresis confirmed our cloned sequence in transformed E. coli cells. Next we cloned the purified 1.1 kb IL8RB sequence into the mammalian expression vector pcDNA 3 (Invitrogen). The fragment will be sequenced to confirm that no errors were introduced by the PCR. The pcDNA 3 vector will then be used to transform human 293 cells, which will express the IL-8RB on the cell surface. Expression will be confirmed by Northern analysis, FACS analysis, and by saturable binding of radiolabeled IL8. I have synthesized eight peptides of approximately 20 amino acids each corresponding to the N-terminal and three extracellular loops of the IL8RB The peptides have all been cleaved from the synthesis resin and deprotected. The next step will be to solubilize the peptides, purify by HPLC and confirm amino acid content. These peptides will be used in competitive binding assays to determine key binding domains of the IL-8RB.